Modified Gene

Figure 81.4 Examples of transgenic modifications. A. A "knockout" consists of deleting the genomic copies of a gene of interest through homologous recombination. A targeting gene carrying a selectable marker, like neomycin resistance, is constructed with ends that have sequence homology to the genomic DNA. Inside the cell recombination between the ends of the targeting gene and the genomic DNA occurs with replacement of part of the gene with the targeting gene. This effectively destroys the function of the endogenous gene. B. A "knock-in" consists of replacing part of the genomic copy of a gene of interest with a modified version of the gene through homologous recombination. A targeting gene carrying the modified version of the gene is constructed with ends that have sequence homology to the genomic DNA. Inside the cell, recombination between the ends of the targeting gene and the genomic DNA occurs with replacement of part of the gene with the modified gene.

regulatory sequences responsible for when and where the gene is expressed. The power of transgenic technology lies in the ability to create animals, where all cells of the animal carry the manipulated DNA so an entire muscle, for example, experiences the effects of the manipulation. Transgenic animals stably transmit the modifications to offspring, providing an essentially unlimited source of animals for study. Additionally, different transgenic animals of the same species can be mated to test the genetic effects of combining two mutations together.

The construction of transgenic animals has been greatly facilitated by the development of transgenic core facilities at many academic institutions. These facilities provide the equipment and technical expertise needed to successfully produce animals, and these services usually are provided on a recharge basis. Consultation with the staff of a core facility, if available, is highly recommended to provide guidance for the experimental strategy.

Muscle atrophy research One challenge involved in studying losses in muscle mass due to aging is the long time required to see the final effects, which can be up to 24 to 27 months (Cartee, 1995). An alternate approach that has been developed is to study muscle atrophy that occurs after hind-limb immobilization, hind-limb suspension, or muscle denervation (Cartee, 1995). These interventions result in profound muscle atrophy over a period of several weeks. Both aging and these interventions demonstrate muscle atrophy, but there are significant differences between the two. The magnitude of muscle mass loss following immobilization, suspension, or denervation is often much greater than the changes seen during aging. Additionally, both type I and type II fibers are affected, whereas type II fibers are preferentially affected by aging. However, research into muscle atrophy has contributed greatly to the understanding of the mechanisms involved in the regulation of muscle mass (McKinnell and Rudnicki, 2004). These studies provide new insights that can now be explored in the context of age-related sarcopenia.

C. elegans

C. elegans is a microscopic organism that has emerged during the last decade to be a powerful genetic system in which to study molecular and biochemical events involved in organismal aging. Under laboratory conditions, worms develop from egg to adult in three days, then reproduce during a four- to five-day reproductive period. The usual total adult life span is 12 to 18 days. Worms develop sarcopenia starting in the postreproductive period at age seven days (Herndon et al., 2002). However, sarcopenia does not develop in a stereotyped fashion in worms of a given age; instead, sarcopenia develops at different ages within a cohort of genetically identical worms grown in culture together. Sarcopenia consists of a reduction in muscle cell size due to loss of cytoplasm and myofibrils. The remaining myofibrils also show a progressive increase in disorganization with loss of the densely packed parallel fibers seen in young worms. In contrast, the nervous system of the worm remains largely intact during aging (Herndon et al., 2002).

The sarcopenia observed with aging has important functional consequences for the affected worms. Beginning at the same time that structural changes occur in the muscles, affected worms show a progressive decline in mobility (Herndon et al., 2002). Young worms demonstrate high levels of activity, which consist of sinusoidal swimming along the surface of an agar plate. The aging worms progress from this initial spontaneous well-coordinated swimming to poorly coordinated swimming only evoked by direct stimulation, which involves touching the worm with a thin platinum wire. Eventually, mobility declines to only minimal movement of the head

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